This application seeks to merge chemical biology, structural biology and pathogen biology (including metabolomics in vitro and infections in mice) for insight into the function of two enzymes in Mtb's intermediary metabolism, lipoamide dehydrogenase (Lpd) and 2-hydroxy-3-oxoadipate (HOA) synthase (HOAS). Lpd participates in 3 enzyme complexes in intermediary metabolism and anti-oxidant defense. Its knockout severely attenuates Mtb in the mouse. We have identified and co-crystallized a species-selective Lpd inhibitor and will identify (and subsequently co-crystallize) others by applying orthogonal high throughput screens: one that tests 2 million compounds against the recombinant enzyme and the other using the hits from the first screen against replicating and non-replicating Mtb. HOAS, which we have crystallized, carries out a newly identified function of the reportedly essential gene Rv1248c, originally mis-annotated as E1 of 1-ketoglutarate (1-KG ) dehydrogenase. This is the first identification of a specific gene product that catalyzes carboligation of 1-KG and glyoxylate, producing HOA and its spontaneous decarboxylation product, hydroxylevulinate. We will replace Rv1248c with a Tet-regulated allele have developed a novel strategy to synthesize HOAS inhibitors from an analog of the cofactor thiamine diphosphate. This work will provide insights about the metabolic constraints faced by Mtb in the mouse, furnish tool compounds for the study of the roles of Lpd and HOAS together with other enzymes, establish if Lpd and HOAS warrant consideration as drug targets, and reveal new aspects of Mtb's intermediary metabolism. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: Most antibiotics target biosynthetic processes and kill replicating bacteria. A major global health need is to eradicate substantially non-replicating subpopulations of Mtb with new chemophores active against new targets. Compounds developed here will inhibit two enzymes that are required by non-replicating Mtb (Lpd) or seem likely to be so (HOAS) and along with metabolomic studies will shed fundamental new light on Mtb's intermediary metabolism.